Cardiomyopathies are myocardial diseases that often lead to cardiac remodeling to compensate for deficiencies in cardiac output (1). Cardiomyopathies are characterized as having systolic dysfunctions (i.e. reduced ejection fraction) in dilated cardiomyopathy or diastolic dysfunctions (i.e. impaired relaxation) in hypertrophic and restrictive cardiomyopathies (2). The regulated thin filament (RTF) is a multi-protein complex responsible for switching cardiac muscle contraction on and off in a calcium dependent manner. Mutations in the genes encoding RTF subunits are often the etiological agents for dilated, hypertrophic and restrictive cardiomyopathies. The RTF is comprised of troponin C (TnC), troponin I (TnI), troponin T (TnT), tropomyosin (Tm) and F-actin. Notably, a hallmark of RTF subunit gene mutations in cardiomyopathies is their ability to alter the calcium sensitivity of cardiac muscle contraction and the morphology of the heart (3). Since multiple forms of cardiomyopathies exist, the identification of new drugs that sensitize (+) or desensitize (-) the calcium sensitivity could potentially reverse these aberrant changes. Moreover, there are no calcium desensitizers in clinical use today. As a result of this HTS campaign, the identification of RTF calcium sensitivity modulators may serve as useful tools for elucidating the roles of these proteins in cardiac muscle contraction and disease (4).

The purpose of this assay is to determine dose response curves for compounds that confirmed activity in a set of previous experiments entitled, "Fluorescence-based biochemical high throughput confirmation assay for inhibitors of the calcium sensitivity of cardiac Regulated Thin Filaments (RTF)" (AID 504635). This biochemical assay employs the visible fluorophore, IANBD (Ex ~ 485 nm; Em ~ 535 nm) attached to troponin C (TnC, the Ca2+ binding subunit of the RTF) to monitor the Ca2+ dependent changes in fluorescence arising from IANBD-labeled RTF. As designed, a decrease in the RTF fluorescence intensity at a fixed calcium concentration and wavelength in response to a test compound will indicate that a change in the RTF calcium sensitivity has occurred. This assay employs an EC70 calcium concentration (1.6 mM). Compounds are tested in triplicate using a 10-point, 1:3 dilution series starting at a nominal concentration of 71.5 uM.

For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Symyx Technologies Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (i.e. 71.5 uM) did not result in greater than 50% inhibition, the IC50 was determined manually as greater than 71.5 uM.

PubChem Activity Outcome and Score:

Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.

Any compound with a percent activity value < 50% at all test concentrations was assigned an activity score of zero. Any compound with a percent activity value >= 50% at any test concentration was assigned an activity score greater than zero. Activity score was then ranked by the potency, with the most potent compounds assigned the highest activity scores.

The PubChem Activity Score range for active compounds is 100-83, and for inactive compounds 77-0.

Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, and compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. This assay monitors the activity of recombinant RTF, which is a complex of 5 proteins. Protein and gene identifiers for one of the components (rabbit fast skeletal alpha actin), was not available in the NCBI database. The MLSMR was unable to provide all samples requested for testing.

Activity Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.

String

2

IC50*

The concentration at which 50 percent of the activity in the inhibitor assay is observed; (IC50) shown in micromolar.

Float

μM

3

LogIC50

Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentration

Float

4

Maximal Response

The maximal or asymptotic response above the baseline as concentration increases without bound.

Float

5

Baseline Response

Adjustable baseline of the curve fit, minimal response value.

Float

6

Inflection Point Concentration

The concentration value for the inflection point of the curve.

Float

μM

7

Hill Slope

The variable 'Hill Slope' describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.

Float

8

Hill S0

Y-min of the curve.

Float

9

Hill Sinf

Y-max of the curve.

Float

10

Hill dS

The range of Y.

Float

11

Chi Square

A measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.

Float

12

Rsquare

This statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.

Float

13

Number of DataPoints

Overall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.